Des-asp{hu 1{b -thr{hu 8 {b angiotensin II as a specific inhibitor for the release of aldosterone

ABSTRACT

A new heptapeptide having specific inhibitory properties for the release of aldosterone from the adrenal cortex has been found. The new compound resembles angiotensin II except for the two terminal amino acids. The N-terminal amino acid (asparatic acid) is absent, while the C-terminus is occupied by L-threonine.

United States Patent 1 Bumpus et al.

[ Nov. 4, 1975 DES-ASW-THR ANGIOTENSIN H AS A SPECIFIC INHIBITOR FOR THERELEASE OF ALDOSTERONE [76] Inventors: Francis Merlin Bumpus, 75

Winterberry Lane, Chagrin Falls, Ohio 44022; Mahesh Chandra Khosla, 7415Warwick Lane, Chesterland, Ohio 44026; Robert Rudolph Smeby, 36801Riviera Road, Willoughby, Ohio 44094 [22] Filed: June 20, 1974 [21]Appl. No.: 481,084

[52] US. Cl. 260/112.5; 424/177 [51] Int. Cl. C07C 103/52; A61K 37/00[58] Field of Search 260/112.5; 424/177 [5 6] References Cited OTHERPUBLICATIONS Allmann et al., Chem. Abst. 76:l22,201f (1972).

Primary ExaminerLewis Gotts Assistant Examiner-Reginald J. SayatAttorney, Agent, or Firm-Paul D. Burgauer; Robert L. Niblack 1 Claim, NoDrawings 1 DES-ASP'-TI-IR ANGIOTENSIN II AS A SPECIFIC INHIBITOR FOR THERELEASE OF ALDOSTERONE DETAILED DESCRIPTION OF THE INVENTION Thisinvention relates to polypeptides. More particularly, it is concernedwith the heptapeptide of the formula:

which is more easily referred to as the heptapeptide chain identifiedas:

L-Arg-L-Val-L-Tyr-L-Ile-L-I-Iis L-ProL-Thr The peptide of this inventionpossesses valuable pharmacological activity. It is capable of inhibitingthe angiotensin II stimulated release of aldosterone from adrenal cortexwhile at the same time inhibiting only poorly the other physiologicaleffects of angiotensin II. Thus, when administered in very small amountsby infusion into peripheral veins of dexamethasone treated bilaterallynephrectomized dogs, the excretion of aldosterone was inhibited. Otherpeptide analogs of angiotensin II which inhibit the pressor effect ofangiotensin II, such as [Ile or [Ala angiotensin II, demonstrate thisaldosterone inhibitory activity weakly or not at all.

By virtue of this selective inhibitory property upon the release ofaldosterone, the peptide of this invention is a valuable agent forcounteracting a number of human diseases in which elevated aldosteroneis a causative factor such as edematous disorders, congestive heartfailure and hypertension.

The heptapeptide of this invention is readily prepared in accordancewith known methods for preparing peptides. Such methods involve thebuilding of a linear chain of amino acids through repetitive amidelinkages employing in such sequential alignment the necessary protectivegroups susceptible to ready removal by conventional cleavage methodswhich do not affect the peptide bonds. The adaptation of such methods tothe peptide of this invention is described in the example below. All theamino acids used in the above structure of Formula I are in theL-configuration. The corresponding chain with D-amino acids does notshow the pharmacological efi'ect described below.

In order to illustrate the method for making the compound of structureI, reference is made to the followingv EXAMPLE A solution of 3.09 g ofBOC-(O-benzyD-threonine and 1.4 ml. of triethylamine in 50 ml. ofethylacetate was added to 10 g. ofchlorometlhylpolystyrene/divinylbenzene (98:2) copolymer of a mesh sizebetween 200 and 400, containing 5.02% chlorine. The mixture was stirredat C. for 36 hours. The esterified polymer was filtered, washed severaltimes, in sequence, with ethanol, dilute acetic acid, water, ethanol andmethanol. The polymer was dried in vacuo over phosphorous pentoxide.Hydrolysis of an aliquot of the polymer and subsequent amino acidanalysis indicated that 0.1 l millimoles of BOC(O-benzyl)-threonine wereesterified per gram of the polymer. Further coupling of the BOC-proline, BOC-N-imidazolebenzyl-histidine, BOC- isoleucine,BOC-(O-benzyl)-tyrosiine, BOC-valine and BOC-nitroarginine in therespective order was carried out by utilizing the action given below foreach amino acid residue. Unless specified, all washings were carried outthree times for three minutes each, first with glacial acetic acid andsecond with methylene chloride. The BOC group was removed by treatmentwith 40% (volume/volume) of trifluoroacetic acid in methylene chloridefor 30 minutes, preceded by a prewash with this reagent for 3 minutes toavoid dilution of the triflu oroacetic acid by the previous methylenechloride wash. The deprotected amino acid polymer ester was washed fivetimes for 3 minutes each with chloroform and the trifluoroacetic saltneutralized by treating the residue for 7 minutes with 10% triethylaminein chloroform, followed by three 3-minute washings with chloroform andmethylene chloride in sequence. The subsequent incoming BOC-amino acidwas added in a 2-fold excess in methylene chloride and the mixture wasstirred for 10 minutes. In the case of BOC-nitroarginine and BOC-benzylhistidine, these materials were first dissolved in dimethylformamide,followed by filtration and mixing the filtrate with one-third volume ofmethylene chloride. Both of these derivatives were used in 3- foldexcesses. Coupling was aided in each instance by the addition of a2-fold excess of dicyclohexylcarbodiimide in methylene chloride andmixing was carried out for 2.5 hours except in the coupling stepsinvolving Arg or His where DCI was used in a 3-fold excess and mixingallowed for eight hours. The polymer-peptide chain was then washedwithDMF followed by a washing with DMF/methylene chloride (llz l and'the coupling step with the BOC-amino, acid and DCl was repeated using amixture of 1: 1 DMF/methylene chloride as the solvent. The polymer chainwas then washed with methanol to remove dicyclohexylurea and finallywashed with DMF. Completeness of each coupling at intermediate stageswas checked by known color reaction tests. All couplings were carriedout at 5C. to avoid racemization', l-hydroxybenzotriazole was used as anadditive to'minimize racemization of histidine during the coupling ofBOC-imidazolebenzyl histidine [see G. C. Windridge and E. C. Jorgensen,J.A.C.S., 93, 6318 7 )1- In the above sequence, the blocked (protected)amino acids were coupled in sequence to the O-benzyl threonine polymer,using BOC-proline, BOC-N- imidazole-benzyl-histidine, BOC-isoleucine,BOC-(O- benzyl))-tyrosine, BOC-valine and BOC-nitroarginine to produce apeptide of the structure of formula I with amino acids 1 7 bound to thepolymer substrate.

The above protected heptapeptide polymer was suspended in approximately100 ml. of freshly distilled trifluoroacetic acid and a slow stream ofhydrogen bromide, prewashed with 10% resorcinol in acetic acid, waspassed through the suspension under anhydrous conditions for about 30minutes with occasional shaking. The suspension was filtered and thepolymer was washed with trifluoroacetic acid. The combined filtrateswere evaporated at room temperature in vacuo. The amorphous powder waswashed with ether, dissolved in a mixture of methanol/acetic acid/water10:1:1 and the solution hydrogenated at 3.5 l g./cm. over 0.5 parts of,palladium black per part of peptide weight for 48 hours with shaking.The product was purified on a 5 X 80 cm. column of Sephadex G 25 (apartially cross-linked dextran gel having an exclusion of molecularweight sizes of 5000, marketed by Pharmacia of Uppsala, Sweden) usingn-butanol/pyridine/- water (1025) as the developing solvent. The averageyield of the product obtained in this manner varied between 40 and 60%based on the millimoles of C-temtinal amino acid esterified onto thepolymer. Fractions in column chromatography were cut without regard foryield to obtain the desired compound in the pure form and no attempt wasmade to rechromatograph the minor fractions for identification purposes.The homogeneity of the compound was determined by thin-layerchromatography in various solvent systems of different pH,electroporesis at pH 1.95 and 8.6 and amino acid analysis, proving thatthe compound is homogeneous with R, 0.45 (n-butanol/acetic acid/water 4:l :5) and R, 0.67 (n-butanol/ethylacetate/acetic acid/water l:l:l:l), R;0.17 (n-butanol/pyridine/water 10125), R 0.52 (n-butanol/aceticacid/water/pyridine 15:3:12: 10) on cellulose thin-layer plates. Thechemical analysis showed that the required amino acids were present inthe expected ratio.

For studying the inhibitory properties for the excretion of aldosterone,male mongrel dogs (20-25 kg) were bilaterally nephrectomized and weretreated with dexamethasone to suppress the secretion ofadrenocorticotropic hormone (ACTH). Arterial pressure was monitoredcontinuously through an indwelling catheter in the femoral artery.Infusates were given into peripheral veins and timed samples of adrenalvenous effluent for steroid determinations were collected from the leftlumboadrenal vein. Angiotensin II was first infused at constant doses of20 ng/kg/min for 15 minutes followed by the heptapeptide of thestructure I. Plasma aldosterone was measured by a radioimmunoassaymethod as described by Farmer et al. (J. Clin. Endocrinol. Metab. 36,460, 1973). The results indicate that the heptapeptide blocked thesecretion of aldosterone at a dose level of 200 ng to 800 ng/kg/min.

Since the above compound is particularly suitable for injection orinfusion, it is particularly valuable that the compound iswater-soluble. A suitable dosage unit can be prepared by simplydissolving the above compound in water or physiological saline at aconcentration of between 50 and 6000 ng/ml. Such a solution can beadministered directly or it can be stored under proper conditions forperiods of several weeks without deterioration, particularly whencombined with l 5% of a preservative such as benzyl alcohol and/or isbuffered to a suitable pH with a nontoxic, pharmaceutically acceptablebuffer. A commonly employed buffer for an injectable solution istris(hydroxymethyl)aminomethane but simple salts such as sodiumphosphate or acetate can be used. Preferably, the vehicle or medium inwhich the compound of formula I is dissolved for an in jectable orinfusable solution is buffered to a pH of 7 to 7.5. a What is claimedis:

1. The heptapeptide L-Ary-L-Val-L-Tyr-L-Ile-L-His- L-Pro-L-Thr.

1. THE HEPATAPEPTIDE L-ARY-L-VAL-L-TYR-L-IIE-L-HIS-L-PROL-THR.